Am. J. Trop. Med. Hyg., 59(6), 1998, pp.

916–921
Copyright q 1998 by The American Society of Tropical Medicine and Hygiene

FIELD STUDY ON THE DISTRIBUTION OF ENTAMOEBA HISTOLYTICA AND

ENTAMOEBA DISPAR IN THE NORTHERN PHILIPPINES AS DETECTED BY THE
POLYMERASE CHAIN REACTION
WINDELL L. RIVERA, HIROSHI TACHIBANA, AND HIROJI KANBARA
Department of Protozoology, Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japan; Department of Infectious
Diseases, Tokai University School of Medicine, Isehara, Kanagawa, Japan

Abstract. We used the polymerase chain reaction (PCR) to study the distribution of Entamoeba histolytica and
E. dispar in 1,872 individuals in 14 communities in the northern Philippines. Here we report a field study using a
DNA extraction protocol from formalin-fixed stool specimens as previously reported. This assay detected 137 stools
(7.318%) containing E. dispar and 18 stools (0.961%) containing E. histolytica. The most affected age group for E.
histolytica/E. dispar infections were those 5–14 years of age. There was no significant difference in the sex distribution
of E. histolytica, while in the case of E. dispar, a higher prevalence was observed in females (9.186%) than in males
(5.731%) (P , 0.01). An apparent clustering of stool-positive cases of E. histolytica and E. dispar was also observed
in the northern part of the study area. The results of this survey demonstrate that E. dispar is highly prevalent in the
communities studied. Moreover, it offers promise for the PCR using DNA extracted from formalin-fixed stools as a
sensitive epidemiologic tool for detecting E. histolytica and E. dispar infections.

The intestinal protozoan parasite Entamoeba histolytica is
the causative agent of human amebiasis. Entamoeba histo-
lytica is responsible for up to 100,000 deaths per year, plac-
ing it second only to malaria in mortality due to protozoan
parasites.1 The infection is common in developing countries
and predominantly affects individuals with poor socioeco-
nomic conditions, nonhygienic practices, and malnutrition.
A number of epidemiologic studies of E. histolytica in-
fection were performed before the clear distinction of two
separate species, E. dispar and E. histolytica, was estab-
lished.2 Because they are morphologically indistinguishable,
studies based on stool surveys have raised the question on
the validity of most of these studies. Moreover, seroepide-
miologic studies that were carried out in a number of en-
demic countries3–8 usually reflected the seroprevalence of the
disease even in the near past. Also, the major problem with
current serologic test results is that they remain positive for
years after an episode of amebiasis. To date, the standard-
ization of antigen preparation and the relative measurement
of positive cut-off antibody titers for use in serodiagnosis
still pose a problem.
In recent years, a number of methods such as isoenzyme
typing,9 DNA probes for the polymerase chain reaction
(PCR) and hybridization,10–13 restriction pattern analysis,14–19
and monoclonal antibodies20–24 have been developed for the
clear distinction of the two species. Clearly, accurate diag-
nostic tools are required for the clinical and public health
management of the disease. The use of a stool ELISA has
recently been shown to be useful in routine diagnostic pro-
cedure and epidemiologic studies.25,26 However, a compara-
tive study on the use of the ELISA and PCR for the detection
of E. histolytica and E. dispar indicated that the PCR was
more advantageous than the ELISA in epidemiologic stud-
ies.27 It is therefore important to assess the usefulness of the
PCR in accumulating data on the prevalence of E. histolytica
and E. dispar in the field that are more accurate.
We previously reported that a successful extraction of
DNA from formalin-fixed stool samples for the PCR is a
useful tool for rapid diagnosis of both E. histolytica and E.
dispar infections.28 Here we report the application of this
method to document the prevalence of the two species in a
number of communities in the northern Philippines. Our pre-
liminary hospital-based studies showed that amebic diarrheal
diseases in Baguio City and nearby communities in the
northern Philippines are frequent; however, their prevalence
and distribution are not well-characterized. We therefore
conducted a large-scale field study to determine the distri-
bution of E. histolytica and E. dispar in these communities.
MATERIALS AND METHODS
Study sites. The study was conducted during the months
of December 1995, January and December 1996, and Jan-
uary 1997 in the communities (locally called barangays)
within and nearby Baguio City, The Philippines (Figure 1).
This study area belongs to the Cordillera Autonomous Re-
gion and is the highest city in The Philippines, with an av-
erage altitude of 1,500 meters above sea level. Three major
land forms characterize the relief features of the city: valley,
plateau, and steep slopes. Throughout the year, the weather
is cool but is coolest during the months of December to
February with an average temperature of 188C.
Housing is inadequate in the city, given a rapid population
increase and inadequate area for settlement expansion aside
from poor economic conditions. Ninety-nine percent of the
total number of households in the city have access to potable
water. Only 0.95% of the total population have doubtful wa-
ter sources, most of which are places where water pipes are
inaccessible in view of the high elevation of the city.
Collection of samples. The day before the scheduled sam-
ple collection, the research team visited the randomly se-
lected households, explained the purpose of the study and
the requirements for sample collection, and distributed spec-
imen cups. Particular care was taken in explaining the need
to label the specimen cups with name, age, and sex and to
avoid sample contact with soil and with samples from other
individuals. Informed consent was obtained from all individ-
uals. This protocol was approved by the Ethical Committees
for Human Studies of the Institute of Tropical Medicine at
Nagasaki University and Baguio General Hospital and Med-

916
 E. HISTOLYTICA AND E. DISPAR IN THE NORTHERN PHILIPPINES 917

FIGURE 1. Map of Baguio City, The Philippines, showing the study areas.

ical Center (Baguio City, The Philippines). The samples
were then collected the next morning and immediately trans-
ported to the laboratory. Each sample was macroscopically
inspected for its consistency and for the presence of soil
contamination, blood, or mucus. Routine microscopic diag-
nosis of parasites was then performed.
Microscopic examination of parasites. The presence of
parasites was determined by microscopic examination of
fresh stools and formalin-ether concentrated specimens.
Genomic DNA extraction and the PCR. Genomic DNA
was extracted from cysts present in formalin-fixed stool
specimens as previously described28 with slight modifica-
tions. Briefly, the pellet resulting from the sedimentation
procedure was washed four times with phosphate-buffered
saline. The pellet from the last wash was resuspended in TE
buffer (100 mM Tris, pH 8.0, and 25mM EDTA) and sub-
jected to freezing (using dry ice and ethanol for 5 min) and
thawing (at 378C in a water bath for 2 min) six times. After
the last treatment, the solution was mixed with 200 ml of
0.2% Triton-X 100 and then heated in 988C water bath for
10 min. The mixture was then incubated with 200 mg/ml of
proteinase K in lysis buffer (100 mM Tris, pH 8.0, 1% so-
dium dodecyl sulfate, 25 mM EDTA) at 808C for 2 hr, and
the DNA was extracted using the routine phenol-chloroform
extraction procedure.29 The PCR was then carried out using
primers specific for E. histolytica and E. dispar (p11 plus
918 RIVERA AND OTHERS

TABLE 1
Prevalence of single and mixed infections of intestinal protozoans and helminths in the study population based on microscopy*

Mixed infections with

Total number of Prevalence E. histolytica/
Species positive cases rate (%) E. dispar E. coli E. nana Blastocystis sp.

Protozoa
Entamoeba histolytica/E. dispar 152 8.119 – 21 (3) 3 (3) 8 (1)
Entamoeba coli 126 6.730 21 (3) – 3 (3) 13 (1)
Endolimax nana 8 0.427 3 (3) 3 (3) – 2 (1)
Blastocystis sp. 83 4.433 8 (1) 13 (1) 2 (1) –
Giardia intestinalis 5 0.267 0 1 (0) 0 1 (0)
Helminths
Ascaris lumbricoides 57 3.044 0 8 (2) 0 15 (2)
Trichuris trichiura 24 1.282 0 6 (4) 0 11 (4)
* Numbers in parentheses represent the total number of mixed infections between species.

p12 and p13 plus p14, respectively) as previously de-
scribed.12
Serology. Eleven serum samples of individuals infected
with E. histolytica and E. dispar and three samples from
healthy controls were collected. The commercially available
Amoebiasis HA-Test (Japan Lyophilization Laboratory, To-
kyo, Japan) and an indirect hemagglutination assay (IHA)
were used to detect antibodies in the serum samples.
Statistical analysis. Prevalence levels were estimated ac-
cording to each of the variables studied. For measuring the
strength of association, we used the Mantel-Haenszel odds
ratio (OR) with a 95% confidence interval (CI).30
RESULTS
Microscopic examinations. The prevalence of all para-
sites found in the sample of individuals is shown in Table
1. In the course of this study, 1,872 fecal samples were ex-
amined. One hundred fifty-two persons were infected with
E. histolytica/E. dispar, resulting in a prevalence of 8.119%.
Few cases of Endolimax nana and Giardia intestinalis were
found, with prevalence rates of 0.427% and 0.267%, respec-
tively. Infection with E. histolytica/E.dispar was found to be
strongly associated with infection by Entamoeba coli. Hel-
minthic eggs of Ascaris lumbricoides (3.044%) and Tri-
churis trichiura (1.282%) were also observed and found to
be associated only with E. coli and Blastocystis sp. but not
with E. histolytica/E. dispar (Table 1).
Polymerase chain reaction. Eighteen of the 152 E. his-
tolytica/E. dispar microscopically positive samples were
identified as E. histolytica using PCR-specific primers. The
remaining 134 samples were found to be E. dispar based on
the PCR. However, the PCR detected three samples from E.
coli-positive samples as E. dispar. Although we found 21 E.
coli-positive samples in close association with E. histolytica/
E. dispar based on microscopy, we failed to identify these
three samples coinfected with E. dispar. Moreover, no mixed
infections of E. histolytica and E. dispar was detected.
Age, sex and geographic distribution of E. histolytica
and E. dispar infections in the study area. The age-specific
prevalence is presented in Table 2. Both E. histolytica and
E. dispar were most common for those 5–14 years of age,
with prevalence rates of 1.509% (OR 5 2.396, 95% CI 5
0.658–8.730) and 9.056% (OR 5 1.467, 95% CI 5 0.915–
2.352), respectively. These prevalence rates did not show a
statistically significant difference when compared with those
of other age groups for either of the two species. However,
analysis of the combined prevalence rates of E. histolytica
and E. dispar in this age group showed that these were sta-
tistically significant (P , 0.05). On the other hand, subjects
less than 1–4 years of age had prevalence rates of 0.635%
for E. histolytica and 6.355% for E. dispar. In those greater
than 45 years of age, the prevalence rates were 0.523% (OR
5 0.823, 95% CI 5 0.085–7.945) for E. histolytica and
6.806% (OR 5 1.076, 95% CI 5 0.548–2.111) for E. dispar.
There was no statistically significant difference between
the prevalence rates of E. histolytica in males (1.086%) and
females (0.813%) while in E. dispar, a higher prevalence
rate was observed in females (9.186%) than in males
(5.731%) (OR 5 1.664, 95% CI 5 1.174–2.359, P , 0.01)
(Table 3).
The study was carried out in 12 communities of Baguio
City and two nearby communities of La Trinidad and Man-
kayan. The map of the study area is shown in Figure 1 and
the prevalence of E. histolytica and E. dispar by geographic

TABLE 2
Age distribution of Entamoeba histolytica– and E. dispar-positive samples as determined by the polymerase chain reaction

Age group No. of Prevalence No. of Prevalence

(years) Sample size E. histolytica rate (%) OR (95% CI)* E. dispar rate (%) OR (95% CI)*

,1–4 472 3 0.635 1.00† 30 6.355 1.00†

5–14 530 8 1.509 2.396 (0.658–8.730) 48 9.056 1.467 (0.915–2.352)
15–24 251 3 1.195 1.891 (0.389–9.205) 18 7.171 1.138 (0.621–2.086)
25–34 258 1 0.387 0.608 (0.064–5.754) 17 6.589 1.039 (0.561–1.924)
35–44 170 2 1.176 1.861 (0.317–10.941) 11 6.470 1.019 (0.499–2.083)
.45 191 1 0.523 0.823 (0.085–7.945) 13 6.806 1.076 (0.548–2.111)
Total 1,872 18 0.961 137 7.318
* Odds ratio (OR) with 95% confidence interval (CI).
† Reference point for comparison with other groups.
 E. HISTOLYTICA AND E. DISPAR IN THE NORTHERN PHILIPPINES 919

TABLE 3
Sex distribution of Entamoeba histolytica– and E. dispar-positive samples as determined by the polymerase chain reaction

No. of Prevalence No. of Prevalence

Sex Sample size E. histolytica rate (%) OR (95% CI)* E. dispar rate (%) OR (95% CI)*

Male 1,012 11 1.086 1.00† 58 5.731 1.00†

Female 860 7 0.813 0.747 (0.289–1.929) 79 9.186 1.664 (1.174–2.359)
Total 1,872 18 0.961 137 7.318
* Odds ratio (OR) with 95% confidence interval (CI).
† Reference point for comparison with other groups.

area is listed in Table 4. Entamoeba histolytica was found
mostly in the northern part of Baguio City (Ambiong,
3.389%; Dreamland, Quirino Hill, 1.526%; Slaughter House,
3.529%) and in the communities of La Trinidad (Pico,
1.652%) and Mankayan (Sapid, 2.439%). Entamoeba dispar,
on the other hand, was widespread in the whole city but
higher prevalence rates were also observed in the northern
communities.
Development of antibodies against E. histolytica.Of the
155 individuals positive for E. histolytica and E. dispar by
the PCR, only 11 agreed to provide serum samples. Using
the commercially-available Amoebiasis HA-Test, we found
three samples reactive to E. histolytica antigen. Stool sam-
ples from the same individuals showed E. histolytica posi-
tivity in the PCR, while the remaining eight samples were
identified as E. dispar. The three serum samples collected
from healthy controls did not show any reactivity to E. his-
tolytica antigen.
DISCUSSION
Infection with E. histolytica is a severe health problem in
many tropical and subtropical areas of the world, especially
in developing countries. In The Philippines, studies on the
prevalence and distribution of E. histolytica are not well-
documented. A previous epidemiologic study using an IHA
showed stool positivity prevalence rates of 2–8% and sero-
positivity rates of 1–13%.4 However, surveys that determine
the prevalence of infection by stool examination of parasites
measure predominantly E. dispar, since this species is more
common, while serologic surveys reflect the incidence of E.
histolytica infection because E. dispar does not show a pos-
itive serologic test result.31
In this study, we sought to determine the prevalence of E.
histolytica and E. dispar with our previously reported tech-
nique that uses genomic DNA extracted directly from for-
malin-fixed specimens for the PCR. To our knowledge, this
is the first report on the distribution of E. histolytica and E.
dispar in The Philippines since the description of the two
species was established. This survey showed prevalence
rates of 0.961% and 7.318% for E. histolytica and E. dispar,
respectively, in the study population. It is interesting to note
that there is an apparent clustering of stool-positive cases of
E. histolytica in the northern part of the study area. Enta-
moeba dispar, on the other hand, was widespread throughout
the study sites except in one community where no cases
were found. However, high prevalence rates for E. dispar
were also found in the northern region. Several factors may
account for the high prevalence rates of protozoan and hel-
minthic parasite. These include urbanization, availability of
potable water, and population density. In this study, intra-
familial infection with E. histolytica was observed in at least

TABLE 4
Geographic distribution of Entamoeba histolytica– and E. dispar-positive samples as determined by the polymerase chain reaction

No. of E. Prevalence No. of Prevalence

Community Sample size histolytica rate (%) OR (95% CI)* E. dispar rate (%) OR (95% CI)*

North
Sapid, Mankayan 123 3 2.439 14 11.382
Pico, La Trinidad 121 2 1.652 17 14.049
Ambiong 118 4 3.389 13 11.016
Dreamland, Quirino Hill 131 2 1.526 11 8.396
Slaughter House 170 6 3.529 10 5.882
Fairview Road 137 0 0 15 10.948
Subtotal 800 17 2.125 1.00† 80 10.000 1.00†
Central
City Camp Lagoon 123 0 0 10 8.130
Lower/Upper QM 163 0 0 12 7.361
Mirador Hill 110 0 0 0 0
San Luis 161 0 0 6 3.726
Campo Sioco 140 0 0 4 2.857
Subtotal 697 0 0 0.032 (0.005–0.194) 32 4.591 0.433 (0.286–0.655)
South
Smokey Mountain 135 1 0.740 11 8.148
Camp 7 128 0 0 9 7.031
Dontogan 112 0 0 5 4.464
Subtotal 375 1 0.266 0.123 (0.023–0.673) 25 6.666 0.643 (0.404–1.022)
Total 1,872 18 0.961 137 7.318
* Odds ratio (OR) with 95% confidence interval (CI).
† Reference point for comparison with other groups.
920 RIVERA AND OTHERS
three households, indicating the possible involvement of hu-
man waste disposal systems, personal hygiene habits, hous-
ing conditions, and food preparation habits. Aside from the
factors mentioned, the apparent clustering in the northern
region may be due to the lack of an adequate water supply
due to the elevated topography of the study area. Water pipes
are inaccessible to the households; thus, they depend on in-
dividuals who collect, deliver, and sell water in containers.
With regard to age, as shown in Table 2, E. histolytica
and E. dispar prevalence rates were particularly high in 5–
14-year-old age group, with an observable decrease in pos-
itivity with age. A possible reason for this is decreased ex-
posure of individuals to the parasite. Seroepidemiologic
studies in endemic areas of Nicaragua,8 Bangladesh,7 and
Brazil32 showed peak prevalences of E. histolytica in a sim-
ilar age group. Although the purpose of this study was the
detection of parasites, a correlation was observed between
the presence of E. histolytica in asymptomatic individuals
and a positive antibody response in a limited number of se-
rum samples assayed by IHA (one of three seropositive sam-
ples from the 5–14-year-old age group). Since only E. his-
tolytica elicits the production of serum antibodies,33 the pos-
sibility of prior exposure of the individuals to E. histolytica
cannot be excluded. However, confirmation of the presence
of parasites by the PCR rules out this problem.
Microscopic examinations revealed the presence of other
intestinal protozoans as well as helminthic parasites in the
study population. A clear association between E. histolytica/
E. dispar and E. coli was observed. However, coinfection of
three additional samples of E. coli with E. dispar were de-
tected only after the PCR. This also shows the reliability of
the PCR in clearly documenting multiple infections among
subjects in epidemiologic studies.
Mixed infections of E. histolytica and E. dispar were not
observed in the samples tested. This observation was also
shown in other studies involving the distribution of E. his-
tolytica and E. dispar in the field.17,28 Although it is not yet
clear if a competitive phenomenon between E. histolytica
and E. dispar is true in vivo, an in vitro study showed that
only a minuscule amount of E. histolytica can ultimately
outgrow E. dispar in culture in a given period of time.34
Thus, the development of an adequate animal model would
be a prerequisite to prove this phenomenon.35
It was also observed in this study that all the E. histoly-
tica-positive individuals were asymptomatic. This is consis-
tent with reports from endemic areas that showed that most
E. histolytica infections are asymptomatic.36,37 Although
three bloody stool samples and a few cases of diarrhea were
observed among the subjects, based on the PCR results, we
could not attribute these to infections with amebae. More-
over, erythrophagocytosis was not observed in bloody stool
samples after microscopic examination. Thus, the presence
of blood and mucus among individuals with diarrhea maybe
due to other factors.17,26
This study shows that a clear potential application of
PCR-based detection of E. histolytica and E. dispar directly
from stools offers the most promise for epidemiologic stud-
ies. An additional benefit is that it can be used to monitor
the efficacy of treatment, which is usually the limitation of
serologic tests because of the persistence of the antibody
response even after successful treatment. Moreover, the use
of the PCR in detecting E. dispar among cyst carriers can
facilitate longitudinal studies to determine the nature of this
nonpathogenic species in the hosts.
In conclusion, our results have demonstrated that E. dis-
par infections are predominant in Baguio City and nearby
communities in the northern Philippines. However, further
studies that include other variables such as socioeconomic
conditions, educational backgrounds, and types of settle-
ments are needed to clearly document the epidemiology of
amebiasis in the northern Philippines.
Acknowledgments: We are indebted to Virginia de Joya for collab-
orative support, and Bernadette Baldo, Bernard Padaco and the staff
of the Department of Clinical Pathology, Baguio General Hospital
and Medical Center, Baguio City, The Philippines for valuable field
assistance and laboratory work. We are also grateful to Sumihisa
Honda for the statistical analyses, and Miki Kinoshita for the ex-
cellent assistance during the preparation of the manuscript.
Financial support: Windell L. Rivera was a recipient of the Japanese
Government Ministry of Education, Science, Sports and Culture
(Monbusho) Ph.D scholarship. This study was supported in part by
the Cooperative Research Grant 1997-9-A-7 of the Institute of Trop-
ical Medicine, Nagasaki University, Japan.
Authors’ addresses: Windell L. Rivera and Hiroji Kanbara, Depart-
ment of Protozoology, Institute of Tropical Medicine, Nagasaki Uni-
versity, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan. Hiroshi Tachi-
bana, Department of Infectious Diseases, Tokai University School
of Medicine, Bohseidai, Isehara, Kanagawa 259-1193, Japan.
Reprint requests: Windell L. Rivera, Department of Protozoology,
Institute of Tropical Medicine, Nagasaki University, 1-12-4 Saka-
moto, Nagasaki 852-8523, Japan.
REFERENCES
1. World Health Organization, 1997. Entamoeba taxonomy. Bull
World Health Organ 75: 291–292.
2. Diamond LS, Clark CG, 1993. A redescription of Entamoeba
histolytica Schaudinn, 1903 (Emended Walker, 1911) sepa-
rating it from Entamoeba dispar Brumpt, 1925. J Eukaryot
Microbiol 40: 340–344.
3. Caballero-Salcedo A, Viveros-Rogel M, Salvatierra B, Tapia-
Conyer R, Sepulveda-Amor J, Gutierrez G, Ortiz-Ortiz L,
1994. Seroepidemiology of amebiasis in Mexico. Am J Trop
Med Hyg 50: 412–419.
4. Cross JH, Basaca-Sevilla V, 1980. Seroepidemiology of ame-
biasis in The Philippines. Phil Soc Microbiol Infect Dis J 9:
21–26.
5. Cross JH, Wheeling C, Banzon T, Basaca-Sevilla V, Sevilla J,
1977. Amoebiasis and intestinal parasitic infections in a pop-
ulation on Cebu Island, The Philippines. Ann Trop Med Par-
asitol 71: 435–441.
6. Gonzalez CR, Isibasi A, Ortiz-Navarrete V, Paniagua J, Garcia
JA, Ramirez A, Salvatierra B, Tapia R, Sepulveda J, Gutierrez
G, Kumate J, 1995. Prevalence of antibodies against Enta-
moeba histolytica in Mexico measured by ELISA. Epidemiol
Infect 115: 535–543.
7. Hossain MM, Ljungstrom I, Glass RI, Lundin L, Stoll BJ, Huldt
G, 1983. Amebiasis and giardiasis in Bangladesh: parasito-
logical and serological studies. Trans R Soc Trop Med Hyg
77: 552–554.
8. Sierra AT, Ruiz LC, Kettis AA, Huldt G, Jonsson J, Schroeder
H, 1992. Amebiasis in Nicaragua: class specific serum anti-
body responses. Arch Med Res 23: 261–264.
9. Sargeaunt PG, Williams JE, Grene JD, 1978. The differentiation
of invasive and non-invasive Entamoeba histolytica by iso-
enzyme electrophoresis. Trans R Soc Trop Med Hyg 75: 519–
521.
10. Bracha R, Diamond LS, Ackers JP, Burchard GD, Mirelman D,
 E. HISTOLYTICA AND E. DISPAR IN THE NORTHERN PHILIPPINES
1990. Differentiation of clinical isolates of Entamoeba his-
tolytica by using specific DNA probes. J Clin Microbiol 28:
680–684.
11. Garfinkel LI, Giladi M, Huber M, Gitler C, Mirelman D, Revel
M, Rozenblatt S, 1989. DNA probes specific for Entamoeba
histolytica possessing pathogenic and nonpathogenic zymo-
demes. Infect Immun 57: 926–931.
12. Tachibana H, Kobayashi S, Takekoshi M, Ihara S, 1991. Dis-
tinguishing pathogenic isolates of Entamoeba histolytica by
polymerase chain reaction. J Infect Dis 164: 825–826.
13. Acuna-Soto R, Samuelson J, De Girolami P, Zarate L, Millan-
Velasco F, Schoolnick G, Wirth D, 1993. Application of the
polymerase chain reaction to the epidemiology of pathogenic
and nonpathogenic Entamoeba histolytica. Am J Trop Med
Hyg 48: 58–70.
14. Clark CG, Diamond LS, 1991. Ribosomal RNA genes of ‘patho-
genic’ and ‘nonpathogenic’ Entamoeba histolytica are dis-
tinct. Mol Biochem Parasitol 49: 297–302.
15. Cruz-Reyes JA, Spice WM, Rehman T, Gisborne E, Ackers JP,
1992. Ribosomal DNA sequences in the differentiation of
pathogenic and nonpathogenic isolates of Entamoeba histo-
lytica. Parasitology 104: 239–246.
16. Tachibana H, Ihara S, Kobayashi S, Kaneda Y, Takeuchi T, Wa-
tanabe Y, 1991. Differences in genomic DNA sequences be-
tween pathogenic and nonpathogenic isolates of Entamoeba
histolytica identified by polymerase chain reaction. J Clin Mi-
crobiol 29: 2234–2239.
17. Tachibana H, Kobayashi S, Paz KC, Aca IS, Tateno S, Ihara S,
1992. Analysis of pathogenicity by restriction-endonuclease
digestion of amplified genomic DNA of Entamoeba histoly-
tica isolated in Pernambuco, Brazil. Parasitol Res 78: 433–
436.
18. Tannich E, Horstmann RD, Knobloch J, Arnold HH, 1989. Ge-
nomic DNA differences between pathogenic and nonpatho-
genic Entamoeba histolytica. Proc Natl Acad Sci USA 86:
5118–5122.
19. Tannich E, Burchard GD, 1991. Differentiation of pathogenic
from nonpathogenic Entamoeba histolytica by restriction
fragment analysis of a single gene amplified in vitro. J Clin
Microbiol 29: 250–255.
20. Petri WA Jr, Jackson TFHG, Gathiram V, Kress K, Saffer LD,
Snodgrass TL, Chapman MD, Keren Z, Mirelman D, 1990.
Pathogenic and nonpathogenic strains of Entamoeba histoly-
tica can be differentiated by monoclonal antibodies to the
galactose-specific adherence lectin. Infect Immun 58: 1802–
1806.
21. Tachibana H, Kobayashi S, Cheng XJ, Hiwatashi E, 1997. Dif-
ferentiation of Entamoeba histolytica from E. dispar facili-
tated by monoclonal antibodies against a 150-kDa surface an-
tigen. Parasitol Res 83: 435–439.
22. Tachibana H, Kobayashi S, Kaneda Y, Takeuchi T, Fujiwara T,
1997. Preparation of a monoclonal antibody specific for En-
921
tamoeba dispar and its ability to distinguish E. dispar from
E. histolytica. Clin Diag Lab Immunol 4: 409–414.
23. Tachibana H, Kobayashi S, Kato Y, Nagakura K, Kaneda Y,
Takeuchi T, 1990. Identification of a pathogenic isolate-spe-
cific 30,000-Mr antigen of Entamoeba histolytica by using a
monoclonal antibody. Infect Immun 58: 955–960.
24. Tachibana H, Kobayashi S, Nagakura K, Kaneda Y, Takeuchi T,
1991. Reactivity of monoclonal antibodies to species-specific
antigens of Entamoeba histolytica. J Protozool 38: 329–334.
25. Haque R, Kress K, Wood S, Jackson TFHG, Lyerly D, Wilkins
T, Petri WA Jr, 1993. Diagnosis of pathogenic Entamoeba
histolytica infection using a stool ELISA based on monoclo-
nal antibodies to the galactose-specific adhesin. J Infect Dis
167: 247–249.
26. Haque R, Faruque ASG, Hahn P, Lyerly DM, Petri WA Jr, 1997.
Entamoeba histolytica and Entamoeba dispar infection in
children in Bangladesh. J Infect Dis 175: 734–736.
27. Mirelman D, Nuchamowitz Y, Stolarsky T, 1997. Comparison
of use of enzyme-linked immunosorbent assay-based kits and
PCR amplification of rRNA genes for the simultaneous de-
tection of Entamoeba histolytica and E. dispar. J Clin Micro-
biol 35: 2405–2407.
28. Rivera WL, Tachibana H, Silva-Tahat MRA, Uemura H, Kan-
bara H, 1996. Differentiation of Entamoeba histolytica and
E. dispar DNA from cysts present in stool specimens by poly-
merase chain reaction: its field application in The Philippines.
Parasitol Res 82: 585–589.
29. Sambrook J, Fritsch EF, Maniatis T, 1989. Molecular Cloning:
A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring
Harbor Laboratory Press.
30. Kahn HA, Sempos CT, 1989. Statistical Methods in Epidemi-
ology. New York: Oxford University Press, 45–71, 85–136.
31. Petri WA Jr, 1996. Recent advances in amebiasis. Crit Rev Clin
Lab Sci 33: 1–37.
32. Braga LL, Lima AAM, Sears CL, Newman RD, Wuhib T, Paiva
CA, Guerrant RL, Mann BJ, 1996. Seroepidemiology of En-
tamoeba histolytica in a slum in northeastern Brazil. Am J
Trop Med Hyg 55: 693–697.
33. Jackson TFHG, Ravdin JI, 1996. Differentiation of Entamoeba
histolytica and Entamoeba dispar infections. Parasitol Today
12: 406–409.
34. Clark CG, Diamond LS, 1993. Entamoeba histolytica: an ex-
planation for the reported conversion of ‘‘nonpathogenic’’
amebae to the ‘‘pathogenic’’ form. Exp Parasitol 77: 456–
460.
35. Clark CG, 1995. Evolution of virulence in Entamoeba histoly-
tica. Trop Dis Bull 92: R147–R155.
36. Jackson TFHG, Gathiram V, Simjee AE, 1985. Seroepidemiol-
ogic study of antibody responses to the zymodemes of En-
tamoeba histolytica. Lancet 1: 716–721.
37. Ravdin JI, Jackson TFHG, Petri WA Jr, Murphy CF, Ungar BLP,
Gathiram V, Skilogiannis J, Simjee AE, 1990. Association of
serum antibodies to adherence lectin with invasive amebiasis
and asymptomatic infection with pathogenic Entamoeba his-
tolytica. J Infect Dis 162: 768–772.